Unlike older children, vaccine-induced immunity among infants wanes after initial immunization and multiple boosters are necessary to establish immunologic memory. Compared to formula feeding, infants who are breast fed have improved duration and magnitude of vaccine responses but the mechanisms by which breast feeding enhances immunologic priming are unknown. Breast feeding's enhancement of immune responses to vaccination range from direct effects on immune development to augmentation of innate immunity by altering commensal gut microbiota. The proposed research tests the hypothesis that breast feeding accelerates B cell priming and immunoglobulin diversity following immunizations at 2 and 4 months of age by modulating the microbial colonization of the infant gut. Studies will focus on the response to the conjugated vaccines against Haemophilus influenza (HIb) and Streptococcus pneumonia [Prevnar 13(R)]. A major goal of this application is to determine mechanisms by which breast feeding impacts neonatal B cell development and antibody affinity maturation. To test our hypothesis, immune responses and gastrointestinal microbe colonization from birth to one year of age will be examined among infants who are exclusively breast fed for at least the first four months of life versus those who are exclusively formula fed. The project has three specific aims: 1) To examine the development of immunoglobulin diversity and frequency of somatic hypermutation by deep sequencing and relating the findings to established measures of vaccine response, including post immunization titers against Haemophilus influenza and pneumococcal polysaccharide serotypes contained in the Prevnar 13(R) vaccine and opsonophagocytic activity. 2) To evaluate the relationship between the infant innate immune response and immunoglobulin diversity by examining the extent of T and B cell maturation, the extent of B cell class switch recombination, levels of pro-inflammatory cytokines, extent of macrophage activation, and capacity of neonatal monocytes/macrophages to produce BAFF and APRIL following activation via Toll-like receptors. 3) To assess the relationship between humoral immune responses to vaccination and gut microbiota between breast fed and formula fed infants. The proposed research applies innovative strategies to address a significant gap in our understanding of human immune development. Results will provide novel measures for the role of infant feeding in development of immunity to vaccines and evidence for informed choices by mothers about the nutritional options for their infants. PUBLIC HEALTH RELEVANCE: Healthy newborns receive over 20 vaccinations in the first year of life, a time of B cell hypo-responsiveness as immunity transitions from fetal tolerance to a phase of immune activation driven by the microbial environment. During this time innate immunity synergizes with the emergence of adaptive immunity to establish the breadth and character of the child's immune response. Breastfeeding is associated with immunologic benefit including more robust responses to vaccination but the mechanism by which it enhances immunity is unknown. This application will examine the relationship between breast feeding, the emergence of the gastrointestinal microbiome, innate immunity, and the development of adaptive immunity to vaccination.